Immunoassay techniques have been known for the last few decades and are now commonly used in medicine for a wide variety of diagnostic purposes to detect target analytes in a biological sample. Immunoassays exploit the highly specific binding of an antibody to its corresponding antigen, wherein the antigen is the target analyte. Typically, quantification of either the antibody or antigen is achieved through some form of labeling such as radio- or fluorescence-labeling. Sandwich immunoassays involve binding the target analyte in the sample to the antibody site (which is frequently bound to a solid support), binding labeled antibody to the captured analyte, and then measuring the amount of bound labeled antibody, wherein the label generates a signal proportional to the concentration of the target analyte inasmuch as labeled antibody does not bind unless the analyte is present in the sample.
Cardiac troponins are sensitive and specific biomarkers of cardiac injury. In particular, cTnT is highly cardiac specific, and it is not present in serum following non-myocardial muscle or other tissue damage. In addition, cTnT has been shown to be a more persistent and sensitive biomarker than others used for diagnosing myocardial infarction. Thus cardiac troponins generally are generally useful for diagnosing acute myocardial ischemia, and cTnT is especially useful. Currently a single assay is available for cTnT detection, from Roche Diagnostics. The assay is reportedly limited to detecting human cTnT from human heart tissue to only 7%. (C. Cobbaert et al, Mv. Ned Tijdschr KIM Chem Labgeneesk 2007; 32:175-8). Additionally, like many others, assays for cTnT are subject to interference heterophilic endogeneous antibodies, which produce false positive results and thus misdiagnosis (G. H. White et al., Clin Chem 2002; 48:201-3; N. Shayanfar et al., Swiss Med Wkly 2008; 138:470). False negatives have also been reported. (See, e.g., Mahalingam M, Ottlinger M E. False-negative qualitative cardiac troponin in a 79-year-old man with myocardial infarction. JAMA 1997; 278:2143-4.J).
Additionally, the known assay for cTnT fails to address the problem of circulating endogenous antibodies, or “autoantibodies”. Autoantibodies have been described for cTnT in apparently healthy blood donors (Adamczyk, M., Brashear, R. J., and Mattingly, P. G. (2009) Clin Chem 55, 1592-3; see also US Patent Application No. US20080102481A1). Other previously described autoantibodies are known to create interference in typical sandwich immunoassays composed of two or more analyte-specific antibodies. For example, cardiac troponin-I reactive autoantibodies may interfere with the measurement of cTnI using conventional midfragment-specific immunoassays. Thus, interference from autoantibodies can also produce erroneous results, particularly near the cut-off values established for clinical diagnoses, thereby increasing the risk of false negative diagnostic results and the risk that individuals will not obtain a timely diagnosis. Additionally, autoantibodies may react with antigens, forming macro-complexes which may extend the circulating half-life of the antigen beyond the typical course of the acute release of the antigen. One approach to addressing the problem of interference from autoantibodies is to choose analyte-specific antibodies that bind to specific epitopes distinct from the analyte epitopes that react with the autoantibodies. Following this general approach, efforts have focused on exploring the use of thousands of different combinations of two, three and even four analyte-specific antibodies to avoid interference from autoantibodies. However, this effort has been largely unsuccessful. It is now evident that autoantibodies against complex protein analytes are likely to be polyclonal within a particular sample, and may be even more diverse among samples from different individuals. Interference from diverse polyclonal autoantibodies may explain the observation that as little as 25% or even less of an analyte protein sequence binds to analyte-specific antibodies, which may in turn explain the lack of success using this approach.
A need therefore exists in the art for improved cTnT immunoassay methods that compensate for interference from various substances that may also be present in a test sample, and in particular for such methods that do so without involving redesign of the analyte detection or capture antibodies.